Lewy Body-like Pathology and Loss of Dopaminergic Neurons in Midbrain Organoids Derived from Familial Parkinson's Disease Patient.

Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson's disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

[Ultrastructure of pathologic deposits and cellular inclusions]. / Ultrastruktur pathologischer Ablagerungen und Zellinklusionen.

Intra- and extracellular depositions and inclusions occur in a wide range of diseases with exogenous (e.g. infectious, environmental and toxic) or endogenous (e.g. genetic, inflammatory, neoplastic and degenerative) aetiology. The noxious agent and the pathogenesis influence the organ of manifestation, the subcellular localisation and the ultrastructural appearance of the depositions. Whereas some of the inclusions like pathogens, foreign material (e.g. asbestos) or microvilli have an almost pathognomonic morphology, other inclusions are present in lower amounts also under normal conditions (e.g. lipid vacuoles and glycogen). Therefore, the interpretation of ultrastructural findings makes a correlation with the histological features and clinical constellation necessary. Auxiliary investigations by electron energy loss spectroscopy (EELS) or electron spectroscopic imaging (ESI) provide additional information about the chemical composition of the material and are therefore especially helpful for the identification of foreign substances. This review focuses on a selection of deposits and inclusions relevant to diagnostic pathology.

Nipah Virus Impairs Autocrine IFN Signaling by Sequestering STAT1 and STAT2 into Inclusion Bodies.

Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes fatal infections in humans. As with most disease-causing viruses, the pathogenic potential of NiV is linked to its ability to block antiviral responses, e.g., by antagonizing IFN signaling through blocking STAT proteins. One of the STAT1/2-binding proteins of NiV is the phosphoprotein (P), but its functional role in IFN antagonism in a full viral context is not well defined. As NiV P is required for genome replication and specifically accumulates in cytosolic inclusion bodies (IBs) of infected cells, we hypothesized that this compartmentalization might play a role in P-mediated IFN antagonism. Supporting this notion, we show here that NiV can inhibit IFN-dependent antiviral signaling via a NiV P-dependent sequestration of STAT1 and STAT2 into viral IBs. Consequently, the phosphorylation/activation and nuclear translocation of STAT proteins in response to IFN is limited, as indicated by the lack of nuclear pSTAT in NiV-infected cells. Blocking autocrine IFN signaling by sequestering STAT proteins in IBs is a not yet described mechanism by which NiV could block antiviral gene expression and provides the first evidence that cytosolic NiV IBs may play a functional role in IFN antagonism.

Oligodendrocytes Prune Axons Containing α-Synuclein Aggregates In Vivo: Lewy Neurites as Precursors of Glial Cytoplasmic Inclusions in Multiple System Atrophy?

α-Synucleinopathies are spreading neurodegenerative disorders characterized by the intracellular accumulation of insoluble aggregates populated by α-Synuclein (α-Syn) fibrils. In Parkinson's disease (PD) and dementia with Lewy bodies, intraneuronal α-Syn aggregates are referred to as Lewy bodies in the somata and as Lewy neurites in the neuronal processes. In multiple system atrophy (MSA) α-Syn aggregates are also found within mature oligodendrocytes (OLs) where they form Glial Cytoplasmic Inclusions (GCIs). However, the origin of GCIs remains enigmatic: (i) mature OLs do not express α-Syn, precluding the seeding and the buildup of inclusions and (ii) the artificial overexpression of α-Syn in OLs of transgenic mice results in a burden of soluble phosphorylated α-Syn but fails to form α-Syn fibrils. In contrast, mass spectrometry of α-Syn fibrillar aggregates from MSA patients points to the neuronal origin of the proteins intimately associated with the fibrils within the GCIs. This suggests that GCIs are preassembled in neurons and only secondarily incorporated into OLs. Interestingly, we recently isolated a synthetic human α-Syn fibril strain (1B fibrils) capable of seeding a type of neuronal inclusion observed early and specifically during MSA. Our goal was thus to investigate whether the neuronal α-Syn pathology seeded by 1B fibrils could eventually be transmitted to OLs to form GCIs in vivo. After confirming that mature OLs did not express α-Syn to detectable levels in the adult mouse brain, a series of mice received unilateral intra-striatal injections of 1B fibrils. The resulting α-Syn pathology was visualized using phospho-S129 α-Syn immunoreactivity (pSyn). We found that even though 1B fibrils were injected unilaterally, many pSyn-positive neuronal somas were present in layer V of the contralateral perirhinal cortex after 6 weeks. This suggested a fast retrograde spread of the pathology along the axons of crossing cortico-striatal neurons. We thus scrutinized the posterior limb of the anterior commissure, i.e., the myelinated interhemispheric tract containing the axons of these neurons: we indeed observed numerous pSyn-positive linear Lewy Neurites oriented parallel to the commissural axis, corresponding to axonal segments filled with aggregated α-Syn, with no obvious signs of OL α-Syn pathology at this stage. After 6 months however, the commissural Lewy neurites were no longer parallel but fragmented, curled up, sometimes squeezed in-between two consecutive OLs in interfascicular strands, or even engulfed inside OL perikarya, thus forming GCIs. We conclude that the 1B fibril strain can rapidly induce an α-Syn pathology typical of MSA in mice, in which the appearance of GCIs results from the pruning of diseased axonal segments containing aggregated α-Syn.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Development of Solubilization and Refolding Buffers.

Overexpression of heterologous protein in prokaryotic host cells, such as Escherichia coli, usually leads to formation of inactive and insoluble aggregates known as inclusion bodies (IBs). Recovery of refolded and functionally bioactive proteins from IBs is a challenging task, and a unique condition (e.g., solubilizing and refolding buffers) for each individual protein should be experimentally obtained. Here, we present a simple protocol for development of solubilizing and refolding buffers for successful recovery of pure bioactive proteins from IBs.

High-Throughput Expression of Inclusion Bodies on an Automated Platform.

In bioprocesses, which target the production of recombinant proteins as inclusion bodies, the upstream process has a decisive influence on the downstream operations, especially regarding cell disruption, inclusion body purity and composition, and refolding yield. Therefore, optimization of the processes in fed-batch mode is a major issue, and screening for strains and process conditions are performed in highly labor, time and cost intensive shake flasks or multiwell plates. Thus, high-throughput experiments performed similar to the industrial operating conditions offer a possibility to develop efficient and robust upstream processes. We present here an automated platform for Escherichia coli fed-batch cultivations in parallelized minibioreactors. The platform allows execution of experiments under multiple conditions while allowing for real-time monitoring of critical process parameters and a controlled fermentation environment. By this, the main factors that affect yields and quality of inclusion bodies can be investigated, speeding up the development process significantly.

Design, Production, and Characterization of Catalytically Active Inclusion Bodies.

Catalytically active inclusion bodies (CatIBs) are promising biologically produced enzyme/protein immobilizates for application in biocatalysis, synthetic chemistry, and biomedicine. CatIB formation is commonly induced by fusion of suitable aggregation-inducing tags to a given target protein. Heterologous production of the fusion protein in turn yields CatIBs. This chapter presents the methodology needed to design, produce, and characterize CatIBs.

Thermoinducible E. coli for Recombinant Protein Production in Inclusion Bodies.

The temperature-inducible λpL/pR-cI857 expression system has been widely used to produce recombinant proteins (RPs), especially when it is necessary to avoid the addition of exogenous materials to induce the expression of recombinant genes, preventing contamination of bioprocesses. The temperature increase favors the formation of inclusion bodies (IBs). The temperature upshift could change the metabolism, productivities, cell viability, IBs architecture, and the host cell proteins inside IBs, affecting downstream to obtain the final product. In this contribution, we focus on the relationship between the bioprocesses using temperature increase as inducer, the heat shock response associated with temperature up-shift, the RP accumulation, and the formation of IBs. Here, we describe how to produce IBs and how culture conditions can modulate the composition and architecture of IBs by modifying the induction temperature in RP production.

Inclusion Body Production in Fed-Batch and Continuous Cultivation.

Various fermentation strategies in industrial biotechnology are applied to produce recombinant target proteins using Escherichia coli. These proteins are often expressed as inclusion bodies (IBs), resulting in a high purity, high stability, and high product titers. In state-of-the-art fed-batch processes, product formation takes place in a short period of time. Sterilization, cleaning, and biomass growth are time consuming steps and reduce the space-time yield. Thus, the interest in establishing continuous cultivations, facilitating higher space-time yields, has been increased in recent years. In this protocol, we provide information and a guide to set-up the production of recombinant proteins in fed-batch, as well as in chemostat continuous cultivations using E. coli.

Inclusion Bodies: Status Quo and Perspectives.

Multiple E. coli cultivations, producing recombinant proteins, lead to the formation of inclusion bodies (IBs). IBs historically were considered as nondesired by-products, due to their time- and cost-intensive purification. Nowadays, many obstacles in IB processing can be overcome. As a consequence, several industrial processes with E. coli favor IB formation over soluble production options due to the high space time yields obtained. Within this chapter, we discuss the state-of-the art biopharmaceutical IB process, review its challenges, highlight the recent developments and perspectives, and also propose alternative solutions, compared to the state-of-the art processing.

Optimization of Inclusion Body Formation and Purification in Multi-well Plates.

Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5'-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.

Infrared Spectroscopy for Structure Analysis of Protein Inclusion Bodies.

Infrared (IR) spectroscopy is a widely used technique for evaluation of protein secondary structure. In this chapter, we focus on the application of this analytical technique for analysis of inclusion bodies. After a general introduction to protein analysis by IR spectroscopy, different approaches for spectra acquisition, data processing, and secondary structure evaluation are presented.

High Pressure Homogenization for Inclusion Body Isolation.

High pressure homogenization (HPH) is a commonly used method for cell lysis of Escherichia coli in order to release intracellularly produced recombinant proteins. For misfolded proteins in E. coli, focus is often put on the development of a suitable solubilization and refolding protocol. However, HPH can be a critical unit operation influencing inclusion body (IB) quality and, subsequently, refolding yields. Here, a protocol for homogenization and IB washing is presented in combination with analytical methods suitable to evaluate these unit operations. The protocol is based on a multivariate approach to identify suitable conditions during HPH. Furthermore, the described workflow is easily scalable and can, therefore, also be used if fixed homogenization conditions are already established.

Intensification of Inclusion Body Processing via Temperature-Based Refolding.

Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.

Using High Pressure and Alkaline pH for Refolding.

The expression of recombinant proteins as insoluble inclusion bodies (IB) has the advantage to separate insoluble aggregates from soluble bacterial molecules, thus obtaining proteins with a high degree of purity. Even aggregated, the proteins in IB often present native-like secondary and tertiary structures, which can be maintained as long as solubilization is carried out in non-denaturing condition. High pressure solubilizes IB by weakening hydrophobic interactions, while alkaline pH solubilizes aggregates by electrostatic repulsion. The combination of high pressure and alkaline pH is effective for IB solubilization at a mild, non-denaturing condition, which is useful for subsequent refolding. Here, we describe the expression of recombinant proteins in Escherichia coli using a rich medium to obtain high expression levels, bacterial lysis, and washing of the IB to obtain products of high purity, and, finally, the solubilization and high yield of refolded proteins using high pressure and alkaline pH.

Production of α-Synuclein Fibrillar-Specific scFv from Inclusion Bodies.

Recombinant antibody fragments such as Fab, scFvs, and diabodies against α-syn have become a viable alternative to the conventional full-length antibodies in immunotherapeutic approaches due to their benefits which include smaller size, higher stability, specificity, and affinity. However, the majority of recombinant antibody fragments typically express as inclusion bodies (IBs) in E. coli, which makes their purification incredibly difficult. Here, we describe a method involving a mild solubilizing protocol followed by slow on-column refolding to purify active single-chain variable fragment (scFv-pF) antibody that can recognize the pathogenic α-syn fibrils.

Refolding of Proteins Expressed as Inclusion Bodies in E. coli.

Microbial-based biotherapeutics that are produced in Escherichia coli (E. coli) can be generated intracellularly in the form of inclusion bodies (IBs) or in soluble active form in periplasmic space or extracellularly. Overexpression of these biotherapeutics in E. coli leads to formation of insoluble aggregates called inclusion bodies. These IBs contain misfolded and inactive form of proteins which need to be refolded to obtain a functionally active form of proteins. Here, we discuss refolding of E. coli-based recombinant human granulocyte colony-stimulating factor (GCSF), expressed as IBs, and highlight some of the key features associated with the refolding kinetic reaction.

The Purification of Heme Peroxidases from Escherichia coli Inclusion Bodies: A Success Story Shown by the Example of Horseradish Peroxidase.

In the following chapter a purification process for recombinant Horseradish peroxidase (HRP) produced in Escherichia coli is described. This enzyme is a secretory plant oxidoreductase belonging to the large peroxidase family III within the peroxidase-catalase superfamily of enzymes. It has high biotechnological significance, however, the isolation of the enzyme from its natural source, the horseradish root, has several shortcomings, which makes the development of a recombinant production strategy interesting. The presented protocol covers all process steps from isolation to the final chromatography step; the enzyme is solubilized from insoluble inclusion bodies, refolded and concentrated to yield a high purity enzyme preparation which is comparable to the commercially available plant-derived HRP. Moreover, we believe that this procedure can also be used to process other peroxidases of family II and III of the plant peroxidase superfamily, as they all share the same relevant features like disulfide bonds and a heme group.

Antimicrobial Applications of Inclusion Bodies.

A broad number of inclusion bodies (IBs) potential uses, including biocatalysis, biocompatible nanomaterials, and nanopills for biomedicine, have been described so far. Recently, it has also been shown that they can also be used as antimicrobial agents. Here, we describe the protocol used to produce and purify IBs with antimicrobial activity at desirable yields and also an optimized and simple methodology to determine the antimicrobial activity of IBs against bacterial strains.